Thursday, July 30, 2015

536 - Nitrogenase-catalyzed Ethane Production and CO-sensitive Hydrogen Evolution from MoFe Proteins Having Amino Acid Substitutions in an α-Subunit FeMo Cofactor-binding Domain

To figure out which parts of the nitrogenase protein are important, this study made very specific mutations to amino acids in the protein in Azotobacter vinelandii to see how they affected the catalysis.

What They Saw
They grew cells, wild-type and mutants, with molybdenum, then extracted and tested their nitrogenase. There was a nifEN knockout strain, a nifDK knockout, and others with specific changes in nifD, sometimes combined with nifN knockout.

None of these had nitrogen-fixing activity. All had just as much dinitrogenase reductase activity as the wild-type; some had more. But regarding acetylene reduction, all nifN knockouts had about zero, but the single-mutation strains all had some, though none nearly as much as the wild-type. They each had more ethane production than the wild-type though, so although total reduction and ethylene production were lower, ethane production was higher.

The temperature stability of mutants wasn't all the same either; some were more sensitive to heat. Lowering the temperature below 30ºC also led to a lower proportion of electron flux going to ethane instead of ethylene (in the mutants that produced ethane). No ethane was seen at any temperature in the wild-type. Though these measurements may not have been reliable, so the trend might not be real.

Then they tried adding carbon monoxide (CO) to inhibit the enzymes. The pattern was the same for each strain (they say), but the amount of total inhibition was different; some were less sensitive than the wild-type, some more, some equal to wild-type.

The FeMo cofactor didn't seem to be different in the mutants; extracting it and using it to restore activity to an apoprotein gave the same results from each strain.

After these results on crude extracts, they purified wild-type nitrogenase and the mutants' most stable enzyme (that replaced the glutamine in NifD position 191 with a lysine). Under acetylene, the wild-type enzyme had about the same electron flux with or without 0.2% CO, but more went to ethylene (vs hydrogen) when CO was absent. 3% CO completely inhibited nitrogen fixation (under nitrogen, obviously), but didn't inhibit hydrogen production: about as much was produced with nitrogen and CO as with argon and CO (or argon without CO). Incidentally, this study gave a NH3 to H2 ratio of 1.4 to 1 in 100% nitrogen, which is somewhat lower than the normal 2 to 1.

With the mutant, there was at least 4x less electron flux overall. With CO absent, most of it went to hydrogen when acetylene was present, but what did go to acetylene produced some ethane and more ethylene (as usual). With argon or nitrogen, it all went to hydrogen. When CO was present, the electron flux seemed even more reduced, but the patterns of product were similar.

What This Means
The 191 glutamine residue seems involved in the catalysis, positioned near the FeMo cofactor active center as it is. I am curious about several things, considering nitrogenase's already interesting catalytic abilities: what would similar studies of the other nitrogenases show? What kind of activity might result from other modifications? And, does this kind of modification allow for the reduction of new substrates, such as carbon monoxide itself? These would be interesting studies, if they haven't been done already. Apparently the vanadium nitrogenase is less sensitive to CO than the Mo version here, and it has already been shown to reduce CO to hydrocarbons, at least in vitro.

The difference in acetylene reduction could be due to different affinities: in the wild-type, a new acetylene replaces an old as soon as it is reduced to ethylene, whereas in a mutant, the ethylene remains long enough to be reduced further to ethane. It seems like this difference is due to a difference in the enzyme itself, rather than the cofactor; so the enzyme itself affects the catalysis (though I guess that's not surprising).


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